Chemical modifications play a crucial role in the regulation of biological processes. Protein function is often modulated by tagging with phosphates, sugars, or lipids, while epigenomic marks on DNA or histones can regulate gene expression up or down. One area that lags behind is the mechanistic understanding of the role of RNA chemical modifications, sometimes referred to as the ?epitranscriptome?. The RNA Modification Database lists more than 60 RNA modifications that occur in eukaryotic cells. Transfer and ribosomal RNA have been shown to be heavily modified, and some of these same modifications also occur in messenger RNA and non-coding RNAs. However, the vast majority of these modifications have not been well-studied in messenger and non-coding RNAs. Even though much about RNA modifications remains to be elucidated, there is emerging evidence that RNA modifications are functionally significant and play important roles in biological processes and diseases in vertebrates. Abnormal RNA modifications have been linked to several human diseases including cancer. Defective transfer RNA modifications are associated with several types of cancer, including bladder, breast, cervical, colorectal, skin, testicular and urothelial cancers. The lack of comprehensive nucleotide modification maps for human tRNAs, and tools for analyzing the modification differences, are significant impediments to obtaining a broader understanding of the regulatory roles of these modifications. This contract aims to address this need by developing methods for mapping tRNA modifications from cancer tissues and cell lines.